ULBP2 Research Articles

Effect of entinostat on NK cell-mediated cytotoxicity against osteosarcoma cells and osteosarcoma lung metastasis

ABSTRACTThere is a crucial need for a new therapeutic approach for osteosarcoma (OS) lung metastasis since this disease remains the main cause of mortality in OS. We previously demonstrated that natural killer (NK) cell therapy has minimal efficacy against OS metastasis. This study determined whether the histone deacetylase inhibitor entinostat could immunosensitize OS cells to NK cell lysis and increases the efficacy of NK cell therapy for OS lung metastasis. Entinostat upregulated ligands for NK cell-activating receptors (major histocompatibility complex [MHC] class I polypeptide-related chain A [MICA] and B [MICB]; UL16 binding proteins 1, 2, 5, and 6; and CD155) on OS cells both in vitro and in vivo and led to more susceptibility to NK cell-mediated cytotoxicity in vitro. Importantly, entinostat did not change NK cell viability, receptor expression, or function within the 24-h treatment. We also demonstrated two potential mechanisms by which entinostat enhanced expression of MICA and MICB on OS cells. Although entinostat upregulated ligands for the NK cell activating receptor on OS lung metastasis, it failed to augment the efficacy of NK cell therapy in our nude mouse human OS lung metastasis model. This can be partly explained by our finding that although the infused NK cells were active and functional and could penetrate into the lungs, they failed to infiltrate into the lung nodules. These challenges regarding cellular immunotherapy against solid tumors may be overcome by combination therapy, such as adding a NK cell-activating cytokine (IL-2 or IL-21).

Abstract 5733: Expression levels of UL16 binding protein 1 and natural killer group 2 member D in patients with gastric cancer

Abstract UL16 binding protein 1 (ULBP1) expressed on the tumor cell surface binds to the natural killer group 2 member D (NKG2D) receptor presenting on natural killer (NK), CD8+ T, and gamma delta T cells. However, the roles of ULBP1 and NKG2D expression and associated immune responses in gastric cancer are unclear. Here, we investigated the relationships between ULBP1 and NKG2D expression and clinical outcomes in patients with gastric cancer. The expression levels of ULBP1 and NKG2D were examined in human gastric cancer cell lines and gastric cancer tissues from 98 patients who underwent surgery from 2004 to 2008. MKN-72 cells expressed ULBP1 with ULBP2, -5, or -6. NKG2D was expressed at a higher level after activation on T cells and NK cells. Among tissue sections positive for NKG2D expression, six cases were positive for CD8 and CD56. In all tissues, NKG2D-expressing cells were typically aCD8+ T cells. Patients with NKG2D expression in tumors had significantly longer overall survival (OS) than patients without NKG2D expression in tumors (p = 0.0217). The longest OS was observed in patients positive for both ULBP1 and NKG2D, whereas the shortest OS was observed in patients negative for both ULBP1 and NKG2D. The interaction between ULBP1 and NKG2D may improve OS in patients with gastric cancer and may have applications in immunotherapy for induction of adaptive immunity in patients with cancer. Moreover, ULBP1 and NKG2D may be useful as prognostic biomarker in gastric cancer. Citation Format: Kiyoshi Yoshimura, Moeko Inoue, Tetsuhiko Asao, Masanori Fuse, Satoshi Wada, Atsuo Kuramasu. Expression levels of UL16 binding protein 1 and natural killer group 2 member D in patients with gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5733. doi:10.1158/1538-7445.AM2017-5733

Distinct expression profile of HCMV encoded miRNAs in plasma from oral lichen planus patients

BackgroundOral lichen planus (OLP) is a T cell-mediated autoimmune disease. The aetiology and molecular mechanisms of OLP remain unclear. Human cytomegalovirus (HCMV) infection is a causal factor in the development of various diseases, but the clinical relevance of HCMV in OLP has not been thoroughly investigated.MethodsIn the present study, we firstly examined twenty-three HCMV-encoded microRNA (miRNA) expression profiles in plasma from training set that including 21 OLP patients and 18 healthy controls using RT-qPCR technology. Dysregulated miRNAs were subsequently confirmed in another larger cohort refereed as validation set consisting of 40 OLP patients and 33 healthy controls. HCMV DNA in peripheral blood leukocytes (PBLs) was also measured in an additional cohort of 13 OLP patients and 12 control subjects. Furthermore, bioinformatics analyses, luciferase reporter assay and western blotting were also performed to predict and verify the direct potential targets of HCMV-encoded miRNAs.ResultsThe RT-qPCR results showed that the plasma levels of five HCMV-encoded miRNAs including hcmv-miR-UL112-3p, hcmv-miR-UL22a-5p, hcmv-miR-UL148d, hcmv-miR-UL36-5p and hcmv-miR-UL59 were significantly increased in OLP patients in both training and validation sets. HCMV DNA in PBLs was also significantly higher in OLP patients than in control subjects. Additionally, by using a combination of luciferase reporter assay and western blotting, we demonstrated that cytomegalovirus UL16-binding protein 1, a molecule that mediates the killing of virus-infected cells by natural killer cells, is a direct target of hcmv-miR-UL59.ConclusionsOur results demonstrate a distinct expression pattern of HCMV-encoded miRNAs in OLP patients, which may provide insight into the relationship between HCMV infection and OLP, and warrants additional study in the diagnosis and aetiology of OLP.

A disease-linked ULBP6 polymorphism inhibits NKG2D-mediated target cell killing by enhancing the stability of NKG2D ligand binding.

NKG2D (natural killer group 2, member D) is an activating receptor found on the surface of immune cells, including natural killer (NK) cells, which regulates innate and adaptive immunity through recognition of the stress-induced ligands ULBP1 (UL16 binding protein 1) to ULBP6 and MICA/B. Similar to class I human leukocyte antigen (HLA), these NKG2D ligands have a major histocompatibility complex-like fold and exhibit pronounced polymorphism, which influences human disease susceptibility. However, whereas class I HLA polymorphisms occur predominantly in the α1α2 groove and affect antigen binding, the effects of most NKG2D ligand polymorphisms are unclear. We studied the molecular and functional consequences of the two major alleles of ULBP6, the most polymorphic ULBP gene, which are associated with autoimmunity and relapse after stem cell transplantation. Surface plasmon resonance and crystallography studies revealed that the arginine-to-leucine polymorphism within ULBP0602 affected the NKG2D-ULBP6 interaction by generating an energetic hotspot. This resulted in an NKG2D-ULBP0602 affinity of 15.5 nM, which is 10- to 1000-fold greater than the affinities of other ULBP-NKG2D interactions and limited NKG2D-mediated activation. In addition, soluble ULBP0602 exhibited high-affinity competitive binding for NKG2D and partially suppressed NKG2D-mediated activation of NK cells by other NKG2D ligands. These effects resulted in a decrease in a range of NKG2D-mediated effector functions. Our results reveal that ULBP polymorphisms affect the strength of human lymphocyte responses to cellular stress signals and may offer opportunities for therapeutic intervention.

Vigilin Regulates the Expression of the Stress-Induced Ligand MICB by Interacting with Its 5′ Untranslated Region

NK cells are part of the innate immune system, and are able to identify and kill hazardous cells. The discrimination between normal and hazardous cells is possible due to an array of inhibitory and activating receptors. NKG2D is one of the prominent activating receptors expressed by all human NK cells. This receptor binds stress-induced ligands, including human MICA, MICB, and UL16-binding proteins 1-6. The interaction between NKG2D and its ligands facilitates the elimination of cells under cellular stress, such as tumor transformation. However, the mechanisms regulating the expression of these ligands are still not well understood. Under normal conditions, the NKG2D ligands were shown to be posttranscriptionally regulated by cellular microRNAs and RNA-binding proteins (RBPs). Thus far, only the 3' untranslated regions (UTRs) of MICA, MICB, and UL16-binding protein 2 were shown to be regulated by RBPs and microRNAs, usually resulting in their downregulation. In this study we investigated whether MICB expression is controlled by RBPs through its 5'UTR. We used an RNA pull-down assay followed by mass spectrometry and identified vigilin, a ubiquitously expressed multifunctional RNA-binding protein. We demonstrated that vigilin binds and negatively regulates MICB expression through its 5'UTR. Additionally, vigilin downregulation in target cells led to a significant increase in NK cell activation against said target cells. Taken together, we have discovered a novel mode of MICB regulation.

NKG2D signaling between human NK cells enhances TACE-mediated TNF-α release

Abstract Natural killer group 2 member D (NKG2D) is a strong NK cell activating receptor, with engagement by ligands triggering granule release and cytokine production from NK cells. The function of NKG2D signaling in NK cells has largely been studied in the context of engagement of the receptor by ligands expressed on the surface of target cells, such as tumor cells. Here we report that upon activation with IL-12, IL-15 and IL-18, human NK cells expressed NKG2D ligands of the UL16 binding protein (ULBP) family on the cell surface. NKG2D-ULBP engagement during homotypic interaction between cytokine-stimulated NK cells increased the activity of the metalloprotease TNF-α-converting enzyme (TACE). This enhanced TACE activity significantly increased the release of TNF-α and ULBP proteins from the surface of the NK cells. These results demonstrate that NKG2D signaling is critical for maximal TNF-α release by NK cells. Further, they are the first to demonstrate a role for NKG2D-ligand interaction via homotypic NK cell contact in NK cell effector function.

Hepatic stellate cell interferes with NK cell regulation of fibrogenesis via curcumin induced senescence of hepatic stellate cell

Hepatic fibrosis, a common scarring response to various forms of chronic liver injury, is a precursor to cirrhosis and liver cancer. During liver fibrosis, hepatic stellate cells (HSCs) initially activate and proliferate, which are responsible for the secretion of extracellular matrix components. However, these cells eventually senesce and are cleared by natural killer (NK) cells. Our previous researches have shown that the natural product curcumin could promote the senescence of activated HSC. In this study, we investigated how NK cells target senescent HSC and assessed the effect of this process on liver fibrosis. We found that senescent HSC induced by curcumin are susceptible to NK cells killing, due to the increased expression of NK cell activating ligand major histocompatibility complex class I chain-related genes A (MICA) and UL16-binding proteins 2 (ULBP2), but not Poliovirus Receptor (PVR). Further studies displayed that the interaction between NK cells and senescent LX2 cells stimulated granule exocytosis. Moreover, the inhibition of granule exocytosis weakened the cytotoxicity of NK cells and promoted the accumulation of senescent LX2 cells. Therefore, these aggregated data indicated that NK cells mediated clearance of senescent LX2 cells and granule exocytosis could play a protective role in the improvement of liver fibrosis.

The TGF-β-induced up-regulation of NKG2DLs requires AKT/GSK-3β-mediated stabilization of SP1.

Natural killer (NK) cells play an important role in preventing cancer development. NK group 2 member D (NKG2D) is an activating receptor expressed in the membrane of NK cells. Tumour cells expressing NKG2DL become susceptible to an immune‐dependent rejection mainly mediated by NK cells. The paradoxical roles of transforming growth factor beta (TGF‐β) in regulation of NKG2DL are presented in many studies, but the mechanism is unclear. In this study, we showed that TGF‐β up‐regulated the expression of NKG2DLs in both PC3 and HepG2 cells. The up‐regulation of NKG2DLs was characterized by increasing the expression of UL16‐binding proteins (ULBPs) 1 and 2. TGF‐β treatment also increased the expression of transcription factor SP1. Knockdown of SP1 significantly attenuated TGF‐β‐induced up‐regulation of NKG2DLs in PC3 and HepG2 cells, suggesting that SP1 plays a key role in TGF‐β‐induced up‐regulation of NKG2DLs. TGF‐β treatment rapidly increased SP1 protein expression while not mRNA level. It might be due to that TGF‐β can elevate SP1 stability by activating PI3K/AKT signalling pathway, subsequently inhibiting GSK‐3β activity and decreasing the association between SP1 and GSK‐3β. Knockdown of GSK‐3β further verified our findings. Taken together, these results revealed that AKT/GSK‐3β‐mediated stabilization of SP1 is required for TGF‐β induced up‐regulation of NKG2DLs. Our study provided valuable evidence for exploring the tumour immune modulation function of TGF‐β.

Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression.

Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I-related chain (MIC) A/B and UL16-binding proteins (ULBP)1-6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.

NK cell activating receptor ligand expression in lymphangioleiomyomatosis is associated with lung function decline.

Lymphangioleiomyomatosis (LAM) is a rare lung disease of women that leads to progressive cyst formation and accelerated loss of pulmonary function. Neoplastic smooth muscle cells from an unknown source metastasize to the lung and drive destructive remodeling. Given the role of NK cells in immune surveillance, we postulated that NK cell activating receptors and their cognate ligands are involved in LAM pathogenesis. We found that ligands for the NKG2D activating receptor UL-16 binding protein 2 (ULBP2) and ULBP3 are localized in cystic LAM lesions and pulmonary nodules. We found elevated soluble serum ULBP2 (mean = 575 pg/ml ± 142) in 50 of 100 subjects and ULBP3 in 30 of 100 (mean = 8,300 pg/ml ± 1,515) subjects. LAM patients had fewer circulating NKG2D+ NK cells and decreased NKG2D surface expression. Lung function decline was associated with soluble NKG2D ligand (sNKG2DL) detection. The greatest rate of decline forced expiratory volume in 1 second (FEV1, -124 ± 30 ml/year) in the 48 months after enrollment (NHLBI LAM Registry) occurred in patients expressing both ULBP2 and ULBP3, whereas patients with undetectable sNKG2DL levels had the lowest rate of FEV1 decline (-32.7 ± 10 ml/year). These data suggest a role for NK cells, sNKG2DL, and the innate immune system in LAM pathogenesis.

1528P - Repeated exposure to cisplatin enhances NK cell-mediated cytotoxicity via up-regulation of NKG2D ligands in non-small cell lung cancer cells

Cisplatin (CDDP) is one of the key drugs for non-small cell lung cancer (NSCLC) and CDDP-resistant mechanisms have been investigated, while the role of immune cells on excluding CDDP-treated cancer cells is poorly understood. Here we demonstrate repeated exposure to CDDP enhances NK group 2 member D (NKG2D) ligands MHC class I-related chain A and B (MICA/B) and UL16 binding protein (ULBP)-2/5/6 in NSCLC cell lines while attenuates them in tumor tissue from patients with NSCLC. A549 cells were exposed to 10 µM of CDDP (A549-CR) at least thrice then possible influences of CDDP exposure on expressions of MICA/B and ULBP-2/5/6 were investigated by flow cytometry. NK cell-mediated cytotoxicity against A549-CR cells was assessed by LDH release assay and compared with original A549 cells. The expressions of NKG2D ligands in tissue samples from NSCLC patients who received CDDP-base chemotherapy were also evaluated using immunohistochemical reactions. Basal expression of MICA/B and ULBP-2/5/6 were clearly upregulated in A549-CR cells compared with original A549 cells. As expected, NK cell-mediated cytotoxicity was enhanced against A549-CR cells compared with A549 cells, and this enhancement depended on NKG2D-NKG2D ligands interaction. On the other hand, the expression of MICA/B and ULBP-2/5/6 were lower in residual tumor than in pre-treated samples from patients with NSCLC. Repeated CDDP exposure enhances the expression of NKG2D ligands in A549 cell line, resulted in enhanced NK cell-mediated cytotoxicity in vitro. On the other hand, repeated CDDP exposure attenuated the expression of NKG2D ligands in patients with NSCLC, suggested that the residual tumor was the result of tumor immunoescape from NK cells.

Alternative Splice Transcripts for MHC Class I–like MICA Encode Novel NKG2D Ligands with Agonist or Antagonist Functions

MHC class I chain-related proteins A and B (MICA and MICB) and UL16-binding proteins are ligands of the activating NKG2D receptor involved in cancer and immune surveillance of infection. Structurally, MICA/B proteins contain an α3 domain, whereas UL16-binding proteins do not. We identified novel alternative splice transcripts for MICA encoding five novel MICA isoforms: MICA-A, -B1, -B2, -C, and -D. Alternative splicing associates with MICA*015 and *017 and results from a point deletion (G) in the 5' splice donor site of MICA intron 4 leading to exon 3 and exon 4 skipping and/or deletions. These changes delete the α3 domain in all isoforms, and the α2 domain in the majority of isoforms (A, B1, C, and D). Endothelial and hematopoietic cells contained endogenous alternative splice transcripts and isoforms. MICA-B1, -B2, and -D bound NKG2D by surface plasmon resonance and were expressed at the cell surface. Functionally, MICA-B2 contains two extracellular domains (α1 and α2) and is a novel potent agonist ligand for NKG2D. We found that MICA-D is a new truncated form of MICA with weak affinity for NKG2D despite lacking α2 and α3 domains. MICA-D may functionally impair NKG2D activation by competing with full-length MICA or MICA-B2 for NKG2D engagement. Our study established NKG2D binding for recombinant MICA-B1 but found no function for this isoform. New truncated MICA isoforms exhibit a range of functions that may drive unexpected immune mechanisms and provide new tools for immunotherapy.

Antitumor effects and mechanisms of DC-CIK biotherapy combined with sorafenib against lung adenocarcinoma cells

Objective To investigate the antitumor effects and the possible mechanisms of dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) in combination with sorafenib on two lung adenocarcinoma cell lines, A549 cells (harboring KRAS gene mutation) and PC-9 cells (harboring EGFR gene mutation). Methods DC and CIK cells were routinely generated in vitro by stimulating PBMCs isolated from lung cancer patients with different cytokines and then co-cultured after a week of culturing. Flow cytometry analysis (FCM) was used to analyze the phenotype of DC-CIK cells after 7 days of co-culturing. The 50% inhibitory concentration (IC50) of sorafenib against tumor cells was detected by MTT assay. The tumor cells were treated with DC-CIK cells alone or in combination with sorafenib. The proliferation of tumor cells was tested by CCK-8 kit and dynamically monitored by real-time cellular analysis (RTCA). Annexin-V/PI staining was used to examine the apoptosis rates in each group. Real-time fluorescent quantitative PCR and FCM were respectively performed to detect the expression of natural killer group 2 member D ligands(NKG2DLs) at mRNA and protein levels after the treatment with sorafenib for 24 h. Results There was no significant difference between the IC50 of sorafenib against A549 and PC-9 cells after a 24-hour exposure (P>0.05). Compared with the DC-CIK biotherapy, treating the tumor cells with DC-CIK cells in combination with sorafenib significantly inhibited the cell proliferation and increased the total apoptosis rates of tumor cells (P<0.05). Moreover, the inhibition rates to tumor cell proliferation were enhanced along with the increase of effect-to-target ratio (E/T). Compared with the single-factor treatment groups, the normalized cell index (NCI) in the combined treatment group was significantly decreased. Blocking NKG2D could abate the inhibitory effect of DC-CIK cells on tumor cell proliferation (P<0.05). The expression of NKG2DLs (including ULBP1, UBLP2 and ULBP3) on tumor cells at mRNA and protein levels were increased to different extent after treating with 5 μmol/L of sorafenib for 24 h. Conclusion There was no significant different between the inhibitory effects of sorafenib on the proliferation of lung adenocarcinoma cancer cells harboring KRAS or EGFR gene mutation. The antitumor effects of DC-CIK cells combined with sorafenib on lung adenocarcinoma cells might be induced by regulating the NKG2D-NKG2DLs pathway and enhancing apoptosis. Moreover, the antitumor effects of the combined treatment were better than those of single-factor treatments. Key words: Lung adenocarcinoma; Sorafenib; Cytokine-induced killer cell; Dendritic cell; Immunotherapy

Chimeric antigen receptor T cell therapy targeting NKG2D ligand.

e14523 Background: Chimeric antigen receptor T cell (CAR-T) therapy is promising immunotherapy for cancer. While CAR-T therapy has shown encouraging results against CD19 in acute lymphoblastic lymphoma and other hematologic malignancies, CAR-T therapy targeting solid tumors is having problem with shortage of appropriate target antigens. NKG2D ligand (NKG2DL) is a ligand of NKG2D, which is expressed mainly on NK and γδ T cells, could be an attractive target of CAR-T therapy. NKG2DL such as MICA, MICB, and ULBP1 (UL16 binding protein 1) to ULBP3 are reported to be expressed on several kinds of tumors. Expression of MICA/MICB on tumor cells are reported to correlate with poor prognosis. Here we demonstrate that co-expressions of ULBP1 in cancer and NKG2D in peripheral blood mononuclear cells are associated with better prognosis in gastric cancer. For further analysis, we are investigating whether NKG2DL is appropriate target and which one among NKG2DL is able to be a target for the CAR-T therapy. Methods: We retrospectively reviewed the ULBP1 expression on tumor cells and the NKG2D expression on tumor infiltrating mononuclear cells by immunohistochemical staining in 98 specimens of surgically resected gastric cancer with cases of MP or deeper invasion depth. We also evaluated the correlation of ULBP1 and/or NKG2D expressions and its clinical outcome. Anti-tag(AT)-CAR-T therapy system which our group has been developing was used for this analysis. AT-CAR gene is transferred to activated T cells. ULBP1 and MICA expression vector was made by pcDNA 3.1. This vector was transfected to the SW480 human colon cancer cell line and A549 human lung cancer cell line by lipofection. Cytotoxicity by AT-CAR-T against tumor cells are evaluated using Terascan-VPC and EVOS. Results: Cases with both ULBP1 and NKG2D positive (n = 12) had better clinical outcome than those with doubly negative (n = 21) (5-year survival rate 90% vs 34%; Log-rank P < 0.05). We successfully set up cytotoxic assay for AT-CAR-T system. We also made the ULBP1 and MICA expression vector and tumor cells lines expressing ULBP1 and MICA in SW480. Conclusions: We are analyzing the cytotoxicity against tumor cell line targeting ULBP1 and/or MICA using CAR-T therapy.

71. Validation of Manufacturing NKG2D Chimeric Antigen Receptor T Cells for a First-In-Human Clinical Trial in AML/MDS and Multiple Myeloma

Canonical CAR-T cells are engineered using constructs encoding a single chain variable fragment derived from a murine or humanized antibody, a costimulatory domain connected by a hinge region, and the signaling domain of CD3 zeta. This restricts T cells transduced with these constructs to recognizing one tumor antigen, such as CD19, and a limited set of cancers. Prior work in murine models demonstrated preclinical efficacy of T cells transduced with a novel activating CAR construct that exploits the ability of NK cells to recognize a variety of tumor antigens. Specifically, a CAR was generated by fusion of native full-length human Natural Killer Group 2D (NKG2D) gene with the human CD3 zeta cytoplasmic signaling domain. The NKG2D receptor in association with natural costimulatory molecule DAP10 creates a unique 6 protein receptor complex enabling NK and CD8+ T cells to kill many cells types via recognition of ligands including MIC-A, MIC-B, and UL-16 binding proteins 1-6. NKG2D-ligands are upregulated in malignancy but protein expression is absent or minor in healthy tissues. Complete remissions and durable CD4+ and CD8+ T-cell memory have been demonstrated in murine models of lymphoma, myeloma and ovarian cancer following adoptive therapy with NKG2D CAR T cells. At the same time, NKG2D CAR T cells significantly alter the tumor microenvironment through cytokine secretion to further promote anti-tumor immunity. This technology is being evaluated clinically in a phase I dose-escalation study (ClinicalTrials. gov NCT02203825) arising through collaboration between academic investigators and commercial sponsors. GMP manufacturing procedures for NKG2D CAR T cells were developed to support this first-in-human trial of NKG2D CAR T cells to assess safety and feasibility in acute myeloid leukemia/myelodysplastic syndrome and multiple myeloma. Following isolation of mononuclear cells, T cells were activated with anti-CD3 mAb and IL-2, subjected to 2 rounds of transduction with SFG retroviral vector containing the NKG2D CAR construct (CM-CS1), and expanded in media containing IL-2. GMP manufacturing procedures were validated using T cells from healthy donors as well as patients with AML and myeloma. Validation studies and initial clinical manufacturing demonstrated consistent viability, robust cell expansion, vector-mediated surface expression of NKG2D on a median of 53.6% CD8+ and 90.3% of CD4+ T cells, consistent viral copy number/cell less than 5, and the absence of replication-competent retrovirus in CM-CS1 T cells. Functionally, CM-CS1 T cells exhibit potent IFN-γ production following exposure to NKG2D ligand-expressing tumor cells. We also demonstrated minimal carry-through of malignant myeloid or plasma cells after deriving autologous CM-CS1 T cells from patients with malignancies. The Phase I trial using CM-CS1 T cells is ongoing, with dose-escalation proceeding as planned. Future goals include application of this novel cell therapy to additional malignant diseases.

Cell Surface Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef Alleles.

HIV-1 Vpu and Nef proteins downregulate cell surface levels of natural killer (NK) cell ligands but functional consequences of individual downregulation events are unclear. We tested how well-conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients. Proviral vpu and nef sequences were amplified from 27 chronic HIV patients, subcloned, and tested for their ability to downregulate cell surface receptors. Cell surface downregulation of CD4, CD317/tetherin, and major histocompatibility complex class 1 that exert biological functions other than NK cell activation were well conserved among patient-derived Vpu and Nef variants. Among NK cell ligands, NK-T-B-antigen, poliovirus receptor, and UL16-binding protein were identified as main targets for Vpu and Nef, the downregulation of which by at least 1 viral protein was highly conserved. NK cell ligands displayed specific sensitivity to Vpu (NK-T-B-antigen) or Nef (poliovirus receptor), and downregulation of cell surface UL16-binding protein was identified as a novel and highly conserved activity of HIV-1 Vpu but not Nef. The conservation of downregulation of major NK cell ligands by either HIV-1 Vpu or Nef suggests an important pathophysiological role of this activity, which may impact the acute but not the chronic phase of HIV infection.

Functional consequences of genetic polymorphisms in the NKG2D receptor signaling pathway and putative gene interactions

NKG2D (NK group 2, member D) is an activating natural killer (NK) receptor, which is expressed on NK and CD8 + T cells. On NK cells, NKG2D elicits cytotoxicity and release of cytokines. On CD8 + T cells, it functions as a co-stimulatory molecule. The receptor recognizes several ligands including the major histocompatibility complex (MHC) class I chain-related molecules A (MICA) and B (MICB) as well as the UL16-binding proteins (ULBP). The diversity of NKG2D ligands is further increased by a high degree of genetic variability of the ligands. Recently, an amino acid exchange from valine to methionine at position 129 in MICA has been found to be associated with the outcome of allogeneic hematopoietic stem cell transplantation (HSCT), and the functional consequences of this specific genetic variation have been elucidated. The clinical associations found after HSCT were explainable by the functional differences of the MICA-129 variants. Herein, we discuss how the genetic polymorphisms of NKG2D ligands and NKG2D itself interact and may affect the outcome of HSCT and the susceptibility to other diseases, which have been associated with polymorphisms in the NKG2D signaling pathway.

Inhibition of MMP activity can restore NKG2D ligand expression in gastric cancer, leading to improved NK cell susceptibility

Natural killer (NK) cells can react with tumor cells through the balance of inhibitory and stimulatory signals between NK cell surface receptors and their ligands, such as MHC class I chain-related A (MICA), MHC class I chain-related B (MICB), and several UL16-binding proteins (ULBPs). In the present study, we evaluated the relationship between NKG2D ligand expression and matrix metalloproteinase (MMP) activity in in vitro culture systems of a panel of gastric cancer cell lines (n=10) and clinical samples (n=102). First, the surface expression of NK group 2 member D (NKG2D) ligands (MICA, MICB, ULBP-2, and ULBP-3) on tumor cells was markedly downregulated on in vitro culture, in parallel to the upregulation of MMPs analyzed by gelatin zymography and gene expression microarray, whereas the transcript levels of NKG2D ligands remained unchanged on in vitro culture. Second, MMP-specific inhibitors could restore the downregulated expression of NKG2D ligands and functionally improve susceptibilities to NK cells in vitro. Third, the production of soluble NKG2D ligands was increased on in vitro culture and was inhibited by MMP-specific inhibitors. Finally, there was a significant inverse correlation between MMP-9 expression and NKG2D ligand expression as analyzed by immunohistochemistry in clinical tumor samples. The present study is a comprehensive study demonstrating that upregulation of MMP activity can induce a downregulation of expression of NKG2D ligands in gastric cancer cells, leading to lower-level susceptibility to NK cells.

A short half‐life of ULBP1 at the cell surface due to internalization and proteosomal degradation

The expression of NKG2D ligands (NKG2D-L) flag stressed cells for immune recognition and destruction. A precise control of the cell surface expression of these proteins is therefore required to ensure an appropriate immune response and it is becoming clear that NKG2D ligand expression is regulated at multiple levels. We now report that the surface stability of the human glycosyl-phosphatidyl-inositol (GPI)-anchored ligand ULBP1 (UL16-binding protein) at the plasma membrane is lower than other ULBP molecules. This difference in stability is due neither to shedding nor to a higher internalization rate of ULBP1 but rather occurs because of a rapid degradation of ULBP1 protein after internalization from the cell surface that is blocked by proteasome inhibition. These data indicate that, in addition to the known transcriptional and post-translational mechanisms, surface expression of human NKG2D-L is also regulated by protein turnover and that the brief residence of ULBP1 could contribute to the fine tuning of immune responses.

Cisplatin enhances NK cells immunotherapy efficacy to suppress HCC progression via altering the androgen receptor (AR)-ULBP2 signals

The aim of this study is to investigate the influence of cisplatin on the efficacy of natural killer (NK) cells immunotherapy to suppress HCC progression, and provide valuable information on better application of cisplatin in clinical settings. By using in vitro cell cytotoxicity test and in vivo liver orthotopic xenograft mice model, we identified the role of cisplatin in modulating NK cells cytotoxicity. Luciferase report assay and chromatin immunoprecipitation assay were applied for mechanism dissection. Immunohistochemistry is performed for sample staining. We found cisplatin could enhance the efficacy of NK cells immunotherapy to better suppress HCC progression via altering the androgen receptor (AR)-UL16-binding protein 2 (ULBP2) signals both in vitro and in vivo. Mechanism dissection revealed that cisplatin could suppress AR expression via two distinct ways: increasing miR-34a-5p to suppress AR expression and altering the ubiquitination to accelerate the AR protein degradation. The suppressed AR might then function through up-regulating ULBP2, a natural-killer group 2 member D ligand, to enhance the cytotoxicity of NK cells. Together, these results indicated an unrecognized favoring effect of cisplatin in HCC treatment. By suppressing AR in HCC, cisplatin could up-regulate cytotoxicity of NK cells to better target HCC. This finding may provide a potential new approach to control HCC by combining traditional chemotherapy with immunotherapy.